Ascorbic acid, ascorbate (Asc), also known as vitamin C, is an important antioxidant that plays an essential role in the biosynthesis of numerous bioactive substances. Ascorbate is a water soluble and is essential for human health. Ascorbic acid and its sodium, potassium, and calcium salts are widely used in many fields, serving as an antioxidant in food, animal feed, beverages, cosmetics, and pharmaceutical formulations (Liu, et al., J. Agric. Food Chem. 2016, 64, 371-380). In humans, ascorbate has to be obtained from diet and its deficiency leads to diseases such as scurvy (Padayatty, S J, et al., J Am Coll Nutr. 2003 February; 22(1):18-35). Ascorbate is involved in many biochemical processes including cellular redox regulations and enzymatic reactions, and is implicated in body defence and several different diseases (Arrigoni, O, et al., Biochim. Biophys. Acta, Gen. Subj. 2002, 1569, 1-9). Ascorbate is also very important in plants and is involved in crucial biochemical processes such as photosynthesis and respiration (Smirnoff, N., et al., Crit. Rev. Plant Sci. 2000, 19, 267-290). Due to its antioxidant properties and nutritional value, ascorbate is also a common additive in foods and commercial products. Numerous methods for detecting ascorbic acid have been developed, including titration with an oxidizing agent, electrochemistry, spectrophotometry, chromatography and chemiluminescence, enzymology, and capillary electrophoresis.
Several studies employ electrochemical methods as the detection strategy towards ascorbic acid, which include WO2014041465, CN103604849, CN103399056, CN101587094, CN101059474, U520070074971/EP1606631/WO2004083868, WO2015099546, WO2009021907, U.S. Pat. No. 7,598,546 and CN102564963. For electrochemical detection, an electric current is generated resulting from the oxidation reaction of ascorbic acid at the electrode. The various patents listed above describe different electrode material for the detection. In general, the electrochemical sensor is vulnerable to interference from other redox active chemicals in the biological matrix, because of their similar electric potential.
Other studies describe high-performance liquid chromatography (HPLC) detection and quantification for ascorbate (Novakova and Solich, TrAC Trends in Analytical Chemistry 2008, 27(10), 942-958; and Pastore, et al., Rapid Commun. Mass. Spectrom. 2001, 15(22), 2051-2057). In HPLC, different components in the sample are separated by a column and detected by UV or other detectors. HPLC in general has a large dynamic range and good accuracy. However, analysis time is long (10-45 min for one sample) and expensive instrument with technical skills are required.
Additional investigations describe using optical methods for ascorbate detection. In CN100510704C, gold nanoparticle that displays change in fluorescent properties is used to detect ascorbate. In CN104267013, a mixture of graphene quantum dot and potassium chromate is employed to detect ascorbate, which restores the fluorescence quenched by the salt. Vislisel, et al., Analytical Biochemistry 2007, 365(1), 31-39 describes condensation of dehydroascorbate, an oxidation product of ascorbate, with o-phenylenediamine (OPDA) to form a fluorescent product for the fluorescent detection of ascorbate. Tempol, a nitroxide radical, is used as the oxidant. In JP11326207, the same OPDA condensation strategy is used, but the degree of polarization of fluorescence is measured instead of the emission intensity. Song, et al., Scientific Reports 2015, 5, 14194 also uses a nitroxide radical as the oxidizing partner with ascorbate to control the emission properties of a lanthanide. The fluorophore with the nitroxide radical only has weak fluorescence and ascorbate reduction of the radical restores and enhances the emission of the fluorophore. Ishii, et al., Chem. Commun. 2011, 47, 4932-4934 (“Ishii”), also uses a nitroxide radical as the ascorbate reacting/responding unit which is linked to and alters the emission property of a phthalocyaninatosilicon. Liu, et al., J. Mater. Chem. B 2015, 3, 191-197 describe emission of gold nanoclusters Au8, which was quenched by TEMPO (a nitroxide radical) derivatives upon which addition of ascorbate restores the emission by oxidizing the TEMPO with the vitamin.
U.S. Pat. No. 4,303,409 describes a colorimetric analysis based on metal complexes of different colors. Redox reaction between the metal and ascorbate changes the color of the system and thus allows colorimetric detection of ascorbate. U.S. Pat. No. 3,771,964 describes another colorimetric assay based on a phosphomolybdate salt which changes color in the presence of ascorbate. U.S. Pat. No. 6,153,399 also uses ascorbate oxidase to oxidize ascorbate in the presence of chromogen and peroxidase, and ascorbate concentration is determined by monitoring changes in absorbance.
In DE4304728, ascorbic acid is detected by using a photochemiluminescence (PCL) measurement system. Autooxidation of luminol is inhibited by ascorbate and the lag phase of photochemiluminescence is correlated with the ascorbate concentration.
Au-Yeung, et al., J. Am. Chem. Soc. 2013, 135(40), 15165-15173 (“Au-Yeung”) describes molecular imaging of labile iron(II) pools in living cells. Ascorbic acid is used to increase the pool of labile iron(II) ions. The release of a fluorophore is triggered by the presence of free metal ions and oxygen. Maity, et al., RSC Adv. 2013, 3, 16788-16794 (“Maity”) describes reaction based molecular probes for selective colorimetric and fluororimetric detection of Co(II) and Cu(I). Binding of these metals to the probes triggers the release of a fluorophore. Au-Yeung and Maity detect the presence of metal ions and not ascorbic acid.
Among the ascorbic acid detection methods described above, only few are applicable in the imaging of live cell: Au-Yeung, Maity, Liu and Ishii. Other methods that require sample pretreatment and preparation (e.g. HPLC), the use of additional reagents (e.g. the OPDA method) are not applicable in live biological sample such as living cells. In addition, the use of nitroxide radical as the ascorbate responding functional group in many of the above examples of fluorescent/colorimetric/luminescent methods pose selectivity problems as other radical/paramagnetic species (e.g. transition metals)/reducing agents could also react with the nitroxide radical. Accordingly, there remains a need to develop analytical tools that can efficiently detect ascorbate, and in particular, in live biological samples such as cells.
Therefore, it is an object of the present invention to provide enhanced molecular sensors.
It is another object of the present invention to provide molecular sensors that selectively detect ascorbate or ascorbic acid, and in particular, in commercial samples, live biological samples, or a combination thereof.
It is another object of the present invention to provide metal complexes that selectively detect ascorbate or ascorbic acid.